A Retrospective Medical Record Review to Describe Health Status and Cardiovascular Disease Risk Factors of Bus Drivers in South Africa.

Cardiovascular disease (CVD) is the leading cause of death globally. The occupational challenges of bus drivers may increase their risk of CVD, including developing obesity, hypertension, and diabetes. We evaluated the medical records of 266 bus drivers visiting an occupational medical practice between 2007 and 2017 in Johannesburg, South Africa, to determine the health status of bus drivers and investigate risk factors for CVD, and their impact on the ability to work. The participants were in majority male (99.3%) with a median age of 41.2 years (IQR 35.2); 23.7% were smokers, and 27.1% consumed alcohol. The median body mass index (BMI) was 26.8 m/kg2 (IQR 7.1), with 63.1% of participants having above normal BMI. Smoking, BMI, and hypertension findings were in line with national South African data, but diabetes prevalence was far lower. Undiagnosed hypertension was found in 9.4% of participants, uncontrolled hypertension in 5.6%, and diabetes in 3.0%. Analysis by BMI category found that obesity was significantly associated with increased odds of hypertension. Uncontrolled hypertension was the main reason for being deemed 'unfit to work' (35.3%). Our research highlights the need for more regular screening for hypertension and interventions to address high BMI.

Determinants of adherence to antiretroviral therapy among HIV-positive adults in sub-Saharan Africa: a systematic review.

OBJECTIVE: The rapid scale up of antiretroviral treatment (ART) in sub-Saharan Africa (SSA) has resulted in an increased focus on patient adherence. Non-adherence can lead to drug-resistant HIV caused by failure to achieve maximal viral suppression. Optimal treatment requires the identification of patients at high risk of suboptimal adherence and targeted interventions. The aim of this review was to identify and summarise determinants of adherence to ART among HIV-positive adults. DESIGN: Systematic review of adherence to ART in SSA from January 2002 to October 2014. METHODS: A systematic search was performed in 6 databases (PubMed, Cochrane Library, EMBASE, Web of Science, Popline, Global Health Library) for qualitative and quantitative articles. Risk of bias was assessed. A meta-analysis was conducted for pooled estimates of effect size on adherence determinants. RESULTS: Of the 4052 articles screened, 146 were included for final analysis, reporting on determinants of 161 922 HIV patients with an average adherence score of 72.9%. Main determinants of non-adherence were use of alcohol, male gender, use of traditional/herbal medicine, dissatisfaction with healthcare facility and healthcare workers, depression, discrimination and stigmatisation, and poor social support. Promoters of adherence included counselling and education interventions, memory aids, and active disclosure among people living with HIV. Determinants of health status had conflicting influence on adherence. CONCLUSIONS: The sociodemographic, psychosocial, health status, treatment-related and intervention-related determinants are interlinked and contribute to optimal adherence. Clinics providing ART in SSA should therefore design targeted interventions addressing these determinants to optimise health outcomes.

Stability of HIV-1 Nucleic Acids in Dried Blood Spot Samples for HIV-1 Drug Resistance Genotyping.

Dried blood spots (DBS) are an easy to collect sample-type that can stabilize biological material at ambient temperature for transport and storage, making them ideal for use in resource-limited settings (RLS). We investigated the effect of storage temperature and duration on ability to detect mixed HIV-1 viral RNA populations, and subsequently viral RNA populations in a background of proviral DNA. Part one of the study used DBS samples of whole blood spiked with specific quantities of HIV-1 subtype-B and -C RNA to study mixed virus population detection. Part two used DBS comprising of HIV-1 subtype-B proviral DNA containing U1 cells combined with HIV-1 subtype-C RNA to mimic HIV-1 infected clinical samples as a model system to study the relative stability of HIV-1 RNA and DNA in DBS. Prepared DBS were stored at -20 °C and +30 °C for periods of one day, one, two, and four weeks. Samples were genotyped to determine changes in the detection of mixtures in the sample over time. From two weeks onwards, storage at +30 °C resulted in gradual, time-related reduction in the detection of mixed virus population at log10 VL 4.0 but not at log10 5.0. Proviral DNA and viral RNA were both stable for at least 52 weeks when stored at -20 °C, compared to progressive RNA decay over time at +30 °C. DBS storage conditions and duration had a significant effect on HIV-1 RNA amplification. Our results demonstrate that DBS storage at ambient temperature (+30 °C) should not exceed two weeks, with long-term storage at -20 °C or lower.

Genotype distribution, viral load and clinical characteristics of infants with postnatal or congenital cytomegalovirus infection.

BACKGROUND: Congenital cytomegalovirus infection is a leading cause of long-term sequelae. Cytomegalovirus is also frequently transmitted to preterm infants postnatally, but these infections are mostly asymptomatic. A correlation between cytomegalovirus genotypes and clinical manifestations has been reported previously in infants with congenital infection, but not in preterm infants with postnatal infection. OBJECTIVES: The main objective of this study was to investigate cytomegalovirus genotype distribution in postnatal and congenital cytomegalovirus infection and its association with disease severity. METHODS: Infants admitted to the neonatal intensive care unit of the University Medical Center Utrecht, The Netherlands between 2003-2010 and diagnosed with postnatal or congenital cytomegalovirus infection were included. Classification of cytomegalovirus isolates in genotypes was performed upon amplification and sequencing of the cytomegalovirus UL55 (gB) and UL144 genes. Clinical data, cerebral abnormalities, neurodevelopmental outcome and viral load were studied in relation to genotype distribution. RESULTS: Genotyping results were obtained from 58 preterm infants with postnatal cytomegalovirus infection and 13 infants with congenital cytomegalovirus infection. Postnatal disease was mild in all preterm infants and all had favourable outcome. Infants with congenital infection were significantly more severely affected than infants with postnatal infection. Seventy-seven percent of these infants were symptomatic at birth, 2/13 died and 3/13 developed long-term sequelae (median follow-up 6 (range 2-8) years). The distribution of cytomegalovirus genotypes was comparable for postnatal and congenital infection. UL55 genotype 1 and UL144 genotype 3 were predominant genotypes in both groups. CONCLUSIONS: Distribution of UL55 and UL144 genotypes was similar in asymptomatic postnatal and severe congenital CMV infection suggesting that other factors rather than cytomegalovirus UL55 and UL144 genotype are responsible for the development of severe disease.

Development and evaluation of an affordable real-time qualitative assay for determining HIV-1 virological failure in plasma and dried blood spots.

Virological failure (VF) has been identified as the earliest, most predictive determinant of HIV-1 antiretroviral treatment (ART) failure. Due to the high cost and complexity of virological monitoring, VF assays are rarely performed in resource-limited settings (RLS). Rather, ART failure is determined by clinical monitoring and to a large extent immunological monitoring. This paper describes the development and evaluation of a low-cost, dried blood spot (DBS)-compatible qualitative assay to determine VF, in accordance with current WHO guideline recommendations for therapy switching in RLS. The assay described here is an internally controlled qualitative real-time PCR targeting the conserved long terminal repeat domain of HIV-1. This assay was applied to HIV-1 subtypes A to H and further evaluated on HIV-1 clinical plasma samples from South Africa (n = 191) and Tanzania (n = 42). Field evaluation was performed in Uganda using local clinical plasma samples (n = 176). Furthermore, assay performance was evaluated for DBS. This assay is able to identify VF for all major HIV-1 group M subtypes with equal specificity and has a lower detection limit of 1.00E+03 copies/ml for plasma samples and 5.00E+03 copies/ml for DBS. Comparative testing yielded accurate VF determination for therapy switching in 89% to 96% of samples compared to gold standards. The assay is robust and flexible, allowing for "open platform" applications and producing results comparable to those of commercial assays. Assay design enables application in laboratories that can accommodate real-time PCR equipment, allowing decentralization of testing to some extent. Compatibility with DBS extends access of sampling and thus access to this test to remote settings.

A pragmatic approach to HIV-1 drug resistance determination in resource-limited settings by use of a novel genotyping assay targeting the reverse transcriptase-encoding region only.

In resource-limited settings (RLS), reverse transcriptase (RT) inhibitors form the backbone of first-line treatment regimens. We have developed a simplified HIV-1 drug resistance genotyping assay targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line failures, coupled with minimal cost and labor. The assay comprises a one-step RT-PCR amplification reaction, followed by sequencing using one forward and one reverse primer, generating double-stranded coverage of RT amino acids (aa) 41 to 238. The assay was optimized for all major HIV-1 group M subtypes in plasma and dried blood spot (DBS) samples using a panel of reference viruses for HIV-1 subtypes A to D, F to H, and circulating recombinant form 01_AE (CRF01_AE) and applied to 212 clinical plasma samples and 25 DBS samples from HIV-1-infected individuals from Africa and Europe. The assay was subsequently transferred to Uganda and applied locally on clinical plasma samples. All major HIV-1 subtypes could be detected with an analytical sensitivity of 5.00E+3 RNA copies/ml for plasma and DBS. Application of the assay on 212 clinical samples from African subjects comprising subtypes A to D, F to H (rare), CRF01_AE, and CRF02_AG at a viral load (VL) range of 6.71E+2 to 1.00E+7 (median, 1.48E+5) RNA copies/ml was 94.8% (n = 201) successful. Application on clinical samples in Uganda demonstrated a comparable success rate. Genotyping of clinical DBS samples, all subtype C with a VL range of 1.02E+3 to 4.49E+5 (median, 1.42E+4) RNA copies/ml, was 84.0% successful. The described assay greatly reduces hands-on time and the costs required for genotyping and is ideal for use in RLS, as demonstrated in a reference laboratory in Uganda and its successful application on DBS samples.

Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes.

BACKGROUND: High cost and varying sensitivity for non-B HIV-1 subtypes limits application of current commercial kits for HIV-1 drug resistance genotyping of all major HIV-1 group-M subtypes. OBJECTIVES: Our research aimed to develop and validate an assay specific for all major HIV-1 group-M subtypes for use as an alternative to commercial assays for HIV-1 protease (PR) and reverse transcriptase (RT) drug resistance genotyping. STUDY DESIGN: A nested RT-PCR encompassing the entire PR and RT up to amino acid 321 of HIV-1 was designed to detect HIV-1 group-M subtypes. Primers compatible with group-M subtypes were defined and analytical sensitivity of the assay evaluated using a panel of reference viruses for subtypes A-H and CRF01_AE. The assay was subsequently evaluated on 246 plasma samples from HIV-1 infected individuals harboring various group-M subtypes and viral loads (VLs). RESULTS: All major group-M HIV-1 subtypes were detected with an overall analytical sensitivity of 1.00E+03 RNA copies/ml. Application of the genotyping assay on 246 primarily African clinical samples comprising subtypes A (n=52; 21.7%), B (n=12; 5.0%), C (n=127; 52.9%), D (n=25; 10.4%), CRF01_AE (n=10; 4.2%), and CRF02_AG (n=10; 4.2%), and unassigned variants (n=10; 4.2%), VL range 4.32E+02-8.63E+06 (median 2.66E+04) RNA copies/ml, was ∼98% successful. CONCLUSIONS: A group-M subtype-independent genotyping assay for detection of HIV-1 drug resistance was developed. The described assay can serve as an alternative to commercial assays for HIV-1 drug resistance genotyping in routine diagnostics, and for surveillance and monitoring of drug resistance in resource-limited settings (RLS).

Accumulation of drug resistance and loss of therapeutic options precede commonly used criteria for treatment failure in HIV-1 subtype-C-infected patients.

BACKGROUND: Virological monitoring is essential to identify antiretroviral treatment (ART) failure, but not widely available. Here, accumulation of resistance and consequences for second-line therapy were investigated in African HIV-1 subtype-C-infected patients. METHODS: A total of 836 patients initiated non-nucleoside reverse transcriptase inhibitor (NNRTI)-based ART and received biannual HIV RNA monitoring. When first-line ART was continued despite virological failure (HIV RNA>1,000 copies/ml), genotypic resistance analysis was performed at baseline, first failure (t1), and 6 or 12 months later (t2). Major resistance mutations (IAS), Stanford genotypic sensitivity scores (GSSs) and proportions of patients meeting WHO-defined failure criteria were compared between time points. RESULTS: Most patients (642/836, 77%) reached viral suppression and 145/642 patients (23%) experienced subsequent failure after a median of 18 months. Counselling resulted in virological re-suppression in 27% (39/145) and 40% (58/145) continued first-line ART despite virological failure; 26 patients were included for genotypic analysis.The mean number of major drug resistance mutations per person increased from 2.8 (t1) to 4.3 (t2). Initially, NNRTI-associated mutations (n=47) predominated; only 25 nucleoside reverse transcriptase inhibitor (NRTI)-associated mutations (mainly M184V) were detected. During prolonged viraemia, NRTI resistance increased (n=44, +76%), in particular thymidine analogue mutations (from 4 to 14) and K65R (from 3 to 6). Consequently, GSSs declined from baseline to t1 and t2: from 3.8 to 1.0 to 0.7 (NNRTIs) and from 6.8 to 5.1 to 4.0 (NRTIs). Despite broad resistance, immunological failure was limited at t2. CONCLUSIONS: Rapid accumulation of drug resistance occurred when ART was continued despite virological failure. Treatment options were lost, even when WHO-defined failure criteria were not met. This study calls for wider access to virological monitoring.

Intrapartum single-dose carbamazepine reduces nevirapine levels faster and may decrease resistance after a single dose of nevirapine for perinatal HIV prevention.

BACKGROUND: World Health Organization guidelines recommend zidovudine + lamivudine for 7 days from labor onset in HIV-infected women receiving single-dose nevirapine (sdNVP) to cover prolonged subtherapeutic nevirapine concentrations. Although effective, this is complicated and does not eliminate resistance; alternative strategies could add benefit. METHODS: Antiretroviral-naive HIV-infected pregnant women aged 18-40 years, with CD4 >200 cells per cubic millimeter, able to regularly attend the antenatal clinics in Moshi, Tanzania, were enrolled 1:1 by alternate allocation to receive 200 mg sdNVP alone or in combination with open-label 400-mg single-dose carbamazepine (sdNVP/CBZ) at delivery (ClinicalTrials.gov NCT00294892). The coprimary outcomes were nevirapine plasma concentrations 1 week and nevirapine resistance mutations 6 weeks postpartum. Analyses were based on those still eligible at delivery. RESULTS: Ninety-seven women were assigned to sdNVP and 95 to sdNVP/CBZ during pregnancy, of whom 75 sdNVP and 83 sdNVP/CBZ were still eligible at delivery at study sites. The median (interquartile range) nevirapine plasma concentration was 1.55 (0.88-1.84) mg/L in sdNVP (n = 61) and 1.40 (0.93-1.97) mg/L in sdNVP/CBZ (n = 72) at delivery (P = 0.91), but 1 week later was significantly lower in sdNVP/CBZ [n = 63; 0.09 (0.05-0.20) mg/L] than in sdNVP [n = 52; 0.20 (0.09-0.31) mg/L; rank-sum: P = 0.004] (geometric mean ratio: 0.64, 95% confidence interval: 0.43 to 0.96; P = 0.03). Six weeks postpartum, nevirapine mutations were observed in 11 of 52 (21%) in sdNVP and 6 of 55 (11%) in sdNVP/CBZ (odds ratio = 0.46, 95% confidence interval: 0.16 to 1.34; P = 0.15). CONCLUSIONS: Addition of single-dose carbamazepine to sdNVP at labor onset in HIV-infected, pregnant women did not affect nevirapine plasma concentration at delivery, but significantly reduced it 1 week postpartum, with a trend toward fewer nevirapine resistance mutations.